Stress, anxiety and peripheral benzodiazepine receptor mRNA levels in human lymphocytes

Life Sciences 67 (2000) 2221Ð2231

Stress, anxiety and peripheral benzodiazepine receptor mRNA levels in human lymphocytes Sutisa Nudmamuda, Pilaiwan Siripurkponga, Chittin Chindaduangratanaa, Ponchai Harnyuttanakorna, Pampimol Lotrakulb, Wachira Laarbboonsarpc, Anan Srikiatkhachornd, Naiphinich Kotchabhakdia, Stefano O. Casalottia,* a

Neuro-Behavioural Biology Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya, Nakorn Pathom, Thailand b Child Mental Health Center, Rama VI Road, Bangkok, Thailand c Department of Psychiatry, Chulalongkorn University, Henri Dunan Rd., Bangkok, Thailand d Department of Neurology, Chulalongkorn University, Henri Dunan Rd., Bangkok, Thailand Submitted 10 November 2000; accepted 22 March 2000

Abstract Peripheral benzodiazepine receptor (PBR) mRNA levels were measured in lymphocytes obtained from a cohort of university students and clinically diagnosed anxious patients. The average level of PBR mRNA was decreased in anxious patients compared to a control group. This data conÞrms previously published results, but it also indicates that PBR mRNA levels cannot be used as a sole diagnostic measure of anxiety because the range of the individual PBR mRNA levels of the anxious group overlapped the range of the PBR mRNA levels of the control group. PBR mRNA levels in students following academic examinations were increased in some individuals and decreased in others. In the same cohort of students individual levels of cortisol and prolactin were predominantly increased and decreased respectively. There was no correlation between the individual changes in the hormone levels or PBR mRNA, which suggests that each of these parameters is affected by different environmental and physiological factors. Lymphocyte PBR mRNA measurement is a useful additional methodology for studying human stress responses however, its use in clinical studies would require the elucidation of PBRÕs physiological role. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Peripheral benzodiazepine receptor; University examination; Anxiety; Stress; Cortisol; Prolactin; Lymphocytes

* Corresponding author. Institute of Laryngology and Otology, University College London, 330 GrayÕs Inn Rd., London WC1X 8EE, UK. Tel.: (44)-020-7915-1466; fax (44)-020-7837-9279. E-mail address: [email protected] (S.O. Casalotti) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 0 6 -7

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Introduction The peripheral benzodiazepine receptor (PBR) is an integral membrane protein expressed in many tissues and localized predominantly, but not exclusively, on the outer mitochondrial membrane [1Ð4]. One of the proposed functional roles of PBR is the transport of cholesterol from the outer to the inner mitochondrial membrane, which is the rate-limiting step of steroidogenesis [5]. Although other subunits may be part of the receptor [6Ð8] the 18 kDa subunit of PBR, which has been puriÞed and cloned from rat [9] bovine [10] and human tissue [11], is responsible for both binding to peripheral benzodiazepine drugs and for stimulation of steroid synthesis [2]. Changes in PBR levels have been linked to both the hypothalamo-pituitary-adrenal system [12] and, in the kidney, to the renin-angiotensin system [13]. In lymphocytes, PBR display similar biochemical and pharmacological characteristics to those of PBR found in steroidogenic tissue [14]. However, tissue fractionation studies have shown that a large percentage of lymphocyte PBR is located on the plasma membrane rather than on the mitochondrial membrane [15,16]. Several studies have demonstrated that the density of PBR binding sites is altered by stress stimuli. Acute treatments such as cold swim stress [17], foot-shocks [18] inescapable stress [19] and noise [20] can induce an increase in PBR binding sites while repeated swim stress [21] repeated foot-shocks [18] and food deprivation stress [22] lead to a reduction in the number of PBR binding sites. The mechanism by which these changes occur is not yet clear. Molecular studies of the PBR gene have not identiÞed the nuclear factors involved in PBR gene expression [23,24]. In our previous study, we observed that the changes in PBR gene expression are both tissue and condition dependent. Dexamethasone decreased PBR gene expression in rat adrenal glands but not in other tissues and two experimental stress paradigms had no effect on PBR gene expression in various rat tissues [25]. Tissue speciÞc changes have also recently been reported for inescapable stress followed by avoidance/escape shuttle-box testing [19]. The present study on PBR gene expression in human lymphocytes was prompted by reports that PBR binding sites in platelets were increased in students immediately after academic examination [26], and were depressed both in parachutists following several training jumps and in civilians during war [27,28]. Additionally, clinically deÞned anxious patients had reduced PBR levels [29], which returned to normal following chronic benzodiazepine treatment [30]. During the course of our work, changes in PBR have also been investigated at the gene expression level and a decrease in the level of PBR mRNA in anxious patients has been reported [31]. The aim of this study is to determine whether PBR gene expression changes can be detected both in clinically anxious patients and in normal subjects experiencing a stressful situation and to compare gene expression measurements to the more traditional serum hormone measurements for the study of stress. By studying two diverse groups such as university students and anxious patients utilizing the same technique of PBR gene expression quantitation, this work aims to illustrate that this relatively simple and inexpensive technique could be used both for clinical and human behavioral studies. Methods Subjects First year university students aged 17Ð19 years (n536) from Mahidol University, Salaya, Thailand participated in the Òexamination stress studyÓ. Blood samples (5 ml) were collected

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from the students within 3 hours of the completion of their Þrst major university examination (13:00Ð15:00 hours). They were also physically examined (pulse rate, blood pressure and respiratory rate) and asked to complete a SML90 questionnaire in Thai and to answer additional questions pertinent to their examination experience. Twenty individuals agreed to donate another blood sample two months later at a time when they were not sitting for an examination. Anxious patients (n514) attending out-patient clinics were selected according to the DSM IV criteria for generalized anxiety disorders. Subjects presenting other unrelated symptoms or who had taken tranquilizing drugs in the last two months were excluded. Two different control groups were used in this study. One control group (n570) was age and sex-ratio matched to the student group, and self reported not to be undergoing stressful experiences. This group was utilized to establish the normal range of PBR mRNA level. The second control group (n530) were clinically deÞned non-anxious patients from the same clinic were the anxious patients were recruited. All subjects and controls gave written consent to the study, which was approved by the Thai Ministry of Public Health. Lymphocyte preparation Blood was collected in heparinized tubes, diluted 1:1 in phosphate buffered saline (pH 7.4), layered (10 ml) on Ficoll solution (3 ml) and centrifuged at 1,500 g for 20 min at room temperature. The white ring was collected and centrifuged at 13,500, g for 10 min. The pellet was washed twice and resuspended in phosphate buffer (pH 7.4). An aliquot was used for cell counts and the rest stored at 2708C until used for RNA extraction. Reverse transcriptase-polymerase chain reaction (RT-PCR) ampliÞcation RT-PCR ampliÞcation was carried out as previously described [25]. RNA was extracted from 1 3 106 lymphocytes with Trizol (Gibco Bethesda, USA) and used for RT-PCR. The RT-PCR master mixture contained 13 Taq polymerase buffer, 25 pmol of primers coding for PBR or actin, 0.2 mM of dATP, dCTP, dGTP, 0.04 mM of dTTP and 0.4 mM of ßuorescein12-dUTP (New England Nuclear, Boston, USA), 1.25 Units of Taq DNA polymerase and 4 Units of AMV Reverse Transcriptase (Promega, Madison, USA). Samples were incubated for 30 min at 428C, followed by 27 cycles of 10 sec at 948C; 10 sec at 588C; 30 sec at 728C with a Þnal 10 min extension step at 728C. The primers used were: PBR-forward AGG GTC TCC GCT GGT ACG CC; PBR-reverse: TGG GGC AAC CTC TGA AGC TC; actin-forward: CCC AGA GCA AGA GAG GCA TC; actin-reverse: CTC AGG AGG AGC AAT GAT CT. RT-PCR samples (10 ml) were Þltered by gravity through a nylon membrane in a dot blot apparatus (BioRad, Hercules, USA). The wells were washed with 500 ml TE buffer (1mM EDTA, 10 mM Tris-HCl pH 8.0) by negative pressure. The membrane were washed in 0.5 M NaCl, 0.5 M Tris-HCl (pH 7.5) and exposed to UV light for crosslinking. The membranes were incubated in Blocking Reagent (New England Nuclear, Boston, USA) for 1 hr at room temperature followed by anti-ßuorescein antibodies (1:1000 in Blocking Reagent, New England Nuclear, USA) washed 4 3 5 min with wash buffer, developed with Chemiluminescence Reagent (New England Nuclear, Boston, USA) and immediately exposed to Kodak X-ray Þlm for 15Ð120 min. The Þlm image was scanned and the density of the bands ana-

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lyzed by the NIH program version 1.6 (Wayne Rasband, Freeware http://rsb.info.nih.gov/nihimage/). The quality of RT-PCR products was checked by electrophoresis on 1% agarose gel. The SML 90 questionnaire and hormone assays Each subject answered 90 questions regarding the frequency of a variety of physiological and psychological experiences. The answers are evaluated for 9 major psychological traits and the results compared to the normal range for the Thai population. Cortisol and prolactin were measured from the same batch of blood used for the PBR measurements by routine radioimmunoassay at Siriraj Hospital, Mahidol University, Thailand. Statistical analysis Duplicate RT-PCR assays were performed for each sample. PBR mRNA quantities are expressed as a ratio of PBR RT-PCR values divided by actin RT-PCR values obtained from parallel reactions. Averages 6 standard deviation (SD) are reported. Statistically signiÞcant changes were calculated using the Mann-Whitney Rank Sum Test. For signiÞcance of correlation, PearsonÕs analysis was applied. Results RT-PCR measurement of PBR mRNA in human lymphocytes The amount of RNA extracted from 1 3 106 lymphocytes, which was too small to be quantiÞed by optical density, was deÞned to be 1 RNA unit. The RNA was serially diluted and ampliÞed by 20Ð30 PCR cycles and analyzed by a dot blot assay (Fig. 1B). A linear relationship between initial amount RNA and Þnal PCR product was observed following 27 PCR cycles using between 0.05Ð0.2 units of RNA template (Fig. 1A, 1B). All subsequent reactions were carried out with 0.1 units of RNA. The speciÞcity of the ampliÞcation reaction was tested by ethidium bromide staining of the RT-PCR products. Detection of single bands of the expected size indicates that the RT-PCR reaction was speciÞc (Fig. 1C). Variation of RT-PCR values, expressed as a ratio of PBR over actin, was less than 15% in any of 20 samples for which RNA extraction and RT-PCR ampliÞcation was repeated. Lymphocyte PBR mRNA levels in anxious patients Lymphocyte PBR mRNA levels were measured in 14 out-patients diagnosed as anxious according to DSM-IV criteria for generalized anxiety and in a control group of out patients matched for age and sex-ratio who were not diagnosed as anxious (n530). The average PBR mRNA level of the anxious patients was signiÞcantly lower (p,0.001) than that of the control subjects although not all anxious patients PBR mRNA levels were lower than the average level of the control subjects (Fig. 2). Effect of university examinations on lymphocyte PBR mRNA PBR mRNA levels were also measured in lymphocytes obtained from 36 Þrst-year university students within 3 hours of completing their Þrst mid-term university examination (Òex-

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Fig. 1. RT-PCR measurement of PBR mRNA in human lymphocytes. A: RNA concentration dependence of RTPCR products. 1 unit of RNA corresponds to the amount of RNA extracted from 1 3 106 lymphocytes. 0.1 units were used for subsequent measurements. Each point is the average of two measurements The PCR products are expressed in arbitrary units of intensity of staining of the photogram shown in B. B: Photogram of chemiluminescent DotBlot assay. The Þlm was scanned, the density of the dots was quantiÞed by the NIH-Image software and used to generate the graph in A. C: Ethidium Bromide stained agarose gel of RT-PCR products

amination samplesÓ), and from 20 of the above students two months later (Ònon-examination samplesÓ). Although it was observed that the average PBR mRNA level of these two groups was not signiÞcantly different (p50.427), it was also noted that while the Òexamination samplesÓ PBR mRNA values ranged between 0.64 and 1.26 (arbitrary units), the non examination sample were restricted between 0.66 and 0.91 (arbitrary units). In order to interpret this observation the PBR mRNA levels as well as the cortisol and the prolactin levels were analyzed in the examination and non-examination samples of the 20 students that were followed up. The average values for these parameters indicate that there was no signiÞcant difference between the ÒexaminationÓ and Ònon-examinationÓ PBR values (p50.114) while there was a signiÞcant (p50.010) and a nearly signiÞcant (p50.064) difference in cortisol and prolactin levels, respectively (Table 1). However, analysis of the individual changes of PBR mRNA levels (Fig. 3) suggests that in those subjects in which PBR mRNA level was high at the examination time it was lower at the non-examination time, while in subjects in which PBR mRNA level was low at the examination time it was higher at non-examination time. In contrast most of the cortisol values decreased from examination time to non examination time

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Fig. 2. PBR levels in anxious patients. Scatter plot of PBR mRNA values of anxious patients (n514) and matched controls (n 5 30) expressed as PBR/actin RT-PCR products. Horizontal and vertical lines indicate averages and SD, respectively. ** p,0.001.

and most prolactin level increased from examination time to non-examination time (Fig. 3B,C). It should also be observed that the individuals (identiÞed by numbers, Fig. 3AÐC) that experienced the major changes in PBR mRNA levels are not the same that experienced the major changes in cortisol or prolactin. There was in fact no signiÞcant correlation between cortisol, prolactin and PBR mRNA values among the 20 students. (r , 0.6 PearsonÕs analysis). To further analyze the Þnding that, at the examination time, the PBR mRNA values were increased in some individuals and decreased in others, the examination samples were split Table 1 Effect of examination stress on PBR mRNA, cortisol and prolactin levels at examination and non-examination time PBRa Students All students n520 ÒHigh pbrÓb n55 ÒMedium pbrÓb n511 ÒLow pbrÓb n54

Cortisol (mg/dl)

Prolactin (ng/ml)

Exam

Non-exam

Exam

Non-exam

Exam

Non-exam

0.9060.18 **1.1660.08 0.8760.06 *0.6960.03

0.8260.08 0.8360.08 0.7960.07 0.9060.02

*12.6665.45 12.9462.09 12.9364.89 11.6069.99

8.4463.33 10.6164.93 8.7164.03 7.5060.70

14.2466.60 *13.2062.09 14.6168.44 11.9863.78

18.1167.04 19.4066.55 17.7466.40 17.4569.79

Serum and lymphocytes were separated from blood samples taken from university students within 3 hours of completing their Þrst university examination and two months later at non-examination time. All values are means 6 standard deviation. a PBR mRNA was measured by RT-PCR and is expressed as a ratio of PBR mRNA values over actin mRNA values. b Subjects were assigned to high, low or medium PBR groups according to whether their PBR values at examination time were higher, lower or within the SD values of a control group (n570). This analysis was carried out because the results from Fig. 3 suggest that the examination stress had differentially affected subgroups of students. * SigniÞcantly different from non-examination samples (P,0.05) ** SigniÞcantly different from non-examination samples (P,0.01)

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Fig. 3. Scatter plot of changes of individual levels of PBR mRNA, cortisol and prolactin levels in 20 university students between examination and non-examination time. Diagonal lines join data points of the same individual. Horizontal dotted lines in plot A mark the SD range of the PBR values of a control group (n570) and were used to assign individuals to the high-, medium- and low-PBR groups. PBR levels are expressed as ratio of PBR over actin mRNA levels. The numbers inside each plot identify the individual students and they are arranged in ascending order of their measured value

into Òhigh-PBRÓ, Òmedium-PBRÓ and Òlow-PBRÓ groups according to whether they were above, within or below the standard deviation (SD) range of ÒnormalÓ subjects (dotted lines in Fig. 3A). The ÒnormalÓ subjects were a group of 70 individuals who had a PBR level of 0.89 6 0.14 (mean 6 SD), an age and sex ratio not signiÞcantly different from the examination students and were not undergoing stressful experiences. The Òhigh-PBRÓ group (n55) had a signiÞcantly higher average level of PBR mRNA at examination time compared to its average level at non-examination time. Conversely, the Òlow PBRÓ (n54) group had a significantly lower PBR mRNA level at examination time than at non-examination time while the Òmedium-PBRÓ group (n511) showed no signiÞcant change in PBR level (Table 1). Thus, both Òhigh-PBRÓ and Òlow-PBRÓ groups converged toward average PBR mRNA values. In contrast, the changes in hormonal levels, although not statistically signiÞcant except for prolactin in the Òhigh PBRÓ group, showed a decrease for cortisol and an increase for prolactin at non-examination time for all three PBR groups (Table 1 and Fig. 4).

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Fig. 4. Bar plot of percentage changes of PBR, cortisol and prolactin levels in all 20 followed-up students divided in high- medium- and low-PBR groups. Vertical lines indicate SD. Statistical signiÞcance was calculated using the examination and non-examination values ** p,0.001, * P,0.05.

Physiological tests (pulse rate, blood pressure and respiratory rate) and the SML questionnaire indicated no physical or psychological abnormality, including anxiety, among any of the students. With respect to the studentsÕ high school performance, there was no correlation between the studentsÕ attitude towards the exams or their self-assessed academic preparation with their respective PBR mRNA level. However, it was observed that the Òhigh-PBRÓ group high-school grade point average was signiÞcantly lower than that of the Òmedium-PBRÓ and Òlow-PBRÓ groups. University regulations prevented us from obtaining the exams results. Discussion As we had previously described [25], RT-PCR can be utilized for semi-quantitative measurements of PBR RNA levels provided that the reaction parameters are adjusted in order to achieve a linear relationship between initial amount of RNA and Þnal RT-PCR product. One of the major advantages of using RT-PCR over radioactive ligand binding assay for measuring changes in PBR expression in lymphocytes is that only 5 ml blood samples were collected versus the 30Ð50 ml required for PBR binding studies [26Ð30]. Since only a small fraction of the RNA extracted was used, the assay could be optimized and automated for drop-size blood samples. To our knowledge there has been only one previous report of PBR gene expression changes in human patients [31] and this study is the Þrst to also evaluate an example of occupational stress and to compare the individual changes of PBR mRNA, cortisol and prolactin. Anxious patients had a signiÞcantly lower average PBR mRNA level than the control group. This conÞrms previous reports of PBR levels in anxious patients measured both by ligand binding [29,30] and RT-PCR [31]. In this work we report the individual levels of PBR mRNA and show that not all the anxious patients had a PBR mRNA level below normal. Thus, PBR mRNA level cannot be used as the sole diagnostic tool for anxiety. However, an extended clinical study with a larger cohort may allow to identify environmental and physio-

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logical factors that may have contributed to the decrease of PBR mRNA in certain individuals and consequently help identify the role of PBR in the stress response. Analysis of the individual changes of PBR mRNA measured in students immediately after an university examination, shows that the in some individuals PBR mRNA was increased and in others it was decreased compared to their individual levels at a time away from university examinations. In previous studies of stress-induced changes of PBR levels in both man and experimental animals it has been shown that acute and chronic stress resulted in increased and decreased PBR binding density, respectively [17Ð22,26Ð30]. University examinations are a complex type of stressor involving both acute and chronic elements. It could be speculated that the students with increased PBR mRNA were affected predominantly by the acute stress of sitting for the examination while the students with decreased PBR mRNA were affected by the chronic stress for the preparation for the exam. Those students who showed no change in PBR RNA levels may either have not been sufÞciently stressed by the examination or the chronic and acute effects of stress had canceled each other out. Measurements of PBR one or two days before the exam could have helped to substantiate this hypothesis. One of the main objectives of this study was to compare PBR mRNA measurements to more traditional stress indicators such as serum cortisol and prolactin levels. Among the 20 students that were followed up we observed that at non examination time there was a much wider range in the values of hormone levels than for PBR mRNA. Both cortisol and prolactin are known to be subject to diurnal and biorhythmic variations. The time schedule of the University examination dictated that both examination and non-examination blood samples were taken in the early afternoon which is generally not the time of lowest hormonal ßuctuations. Other factors, such as food consumption and menstrual cycles may have contributed to the variation among the non-examination samples [33]. In contrast PBR mRNA levels were consistently found to be within a relative narrow range among any of the control groups studied. Thus this seems to be an advantage of PBR mRNA measurement over hormone assays. The majority of the students were female, (males n53) which did not allow to carry out gender comparisons. In this study, the majority of cortisol levels were higher, and the majority of the prolactin levels were lower at examination time than their respective values at non-examination time. This is consistent with the current knowledge of stress-induced hormonal changes, however other studies on the effect of examination stress on hormonal changes have reported both signiÞcant and non-signiÞcant changes [26,27,32]. Measurement of serum hormones thus is not a sufÞciently reliable indicator of stress and measurements of additional parameters such as PBR mRNA can aid stress analysis. Additionally, it is interesting to note that there is no correlation among individuals with highest cortisol level, lowest prolactin level or lowest or highest PBR mRNA level. This would suggest that either different stress response mechanism are most prominent in different individuals, or that each of the three parameters studied is affected by a series of independent physiological and environmental factors that could not be accounted for in this study. This further supports the idea that stress studies should be carried out by measuring multiple parameters, including PBR mRNA. The PBR mRNA examination samples were divided into three subgroups according to whether their values were higher lower or within the standard variation range of PBR mRNA values of a large control group. The results show that both the high and the low PBR groups had PBR mRNA levels signiÞcantly different from their respective levels at non-examination time. It would be of interest to extend this study to a much larger cohort and to search for the

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common factors that could be responsible for the observed changes. Such factors may help elucidate the mechanisms of PBR gene expression. In summary, this study has shown that lymphocyte PBR mRNA levels are inßuenced by both chronic anxiety and common stressors. The measurement of PBR mRNA levels can be as practical and economical as that of hormone levels. However, due to the lack of understanding of the precise mechanisms that control PBR gene expression, measurement of PBR mRNA levels is at present time more suited for basic research studies of stress mechanisms rather than for clinical studies. Nonetheless, due to its apparent lower variation at non-stress time, PBR mRNA level measurement could in the future become a useful diagnostic tool. Acknowledgments We thank Dr. Sakol Payim for the use of facilities at the Molecular Biology Institute, Mahidol University, Dr. Sureeporn Punpeng for advice on data analysis and Dr. Supanya Lamsam and Mr. William Longworth for critical review of the manuscript. This work was supported by a Mahidol Research Grant to C.C. References 1. Gavish M, Bachman I, Shoukrun R, Katz Y, Veenman L, Weisinger G, Weizman A. Enigma of the peripheral benzodiazepine receptor. Pharmacological Reviews. 1999; 51:629Ð650. 2. Krueger KE. Molecular and functional properties of mitochondrial benzodiazepine receptors. Biochem Biophys Acta 1995; 1241: 453Ð470. 3. Gavish M, Katz Y, Bar-Ami S, Weizman R. Biochemical physiological and pathological aspects of the peripheral benzodiazepine receptors. Journal of Neurochemistry 1992; 58: 1589Ð1601. 4. Verma A, Snyder SH. Peripheral-type benzodiazepine receptors. Annual Review of Pharmacology and Toxicology 1989; 29: 307Ð322. 5. Papadopoulos V. Peripheral-type benzodiazepine receptor/diazepam binding inhibitor receptor: Biological role in steroidogenic cell function. Endocrinology Reviews 1993; 14: 222Ð240. 6. Blahos J II, Whalin ME, Krueger KE. IdentiÞcation and puriÞcation of a 10-Kilodalton protein associated with mitochondrial benzodiazepine receptors. Journal of Biological Chemistry 1995; 270: 20285Ð20291. 7. McEnery MW, Snowman AM, TriÞletti RR, Snyder SH. Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proceeding of the National Academy of Science USA 1992; 89: 3170Ð3174. 8. Snyder SH, Verma A, TriÞletti RR. The peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. FASEB Journal 1987; 1: 282Ð288. 9. Sprengel R, Werner P, Seeburg PH, Mukhin AP, Santi AG, Grayson DR, Guidotti A, Krueger KE. Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor. Journal of Biological Chemistry 1989; 264: 20415Ð20421. 10. Parola AL, Stump DG, Pepperl DJ, Krueger KE, Regan JW, Laird HE 2d. Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein. Journal of Biological Chemistry 1991; 266: 14082Ð14087. 11. Riond J, Mattei MG, Kaghad M, Dumont X, Guillemot JC, Le Fur G, Caput D, Ferrara P. Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor. European Journal of Biochemistry 1991; 195: 305Ð311. 12. Papadopoulos V, Nowzari FB, Krueger KE. Hormone-stimulated steroidogenesis is coupled to mitochondrial benzodiazepine receptor: tropic hormone action on steroid biosynthesis inhibited by ßunitrazepan. Journal of Biological Chemistry 1991; 266: 1Ð6.

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